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The objective of this study was to identify and characterize naturally-occurring communities of Bacteria and Fungi fouling the surfaces of concrete structures in Georgia, USA, through the use of culture-independent and culture-dependent approaches. Genomic DNA was extracted and ribosomal RNA genes were PCR amplified from 4 biofouled sites located in or around the cities of Atlanta, Gainesville, LaGrange, and Savannah. Bacterial and fungal community composition was determined by phylogenetic analysis. Molecular analysis revealed five bacterial phyla, and representatives of the phylum Cyanobacteria and the classes Betaproteobacteria and Gammaproteobacteria dominated the bacterial clone libraries. Fungal clone libraries showed the dominant phylotypes to be most closely related to Alternaria, Cladosporium, Epicoccum and Udeniomyces. Phylogenetically distinct microbial populations were present at each of the biofouled sites. In addition, cultured isolates were obtained from sites and tested for their ability to foul concrete of varied compositions under laboratory-controlled conditions. Biofouling tests revealed that fungal isolates obtained from the field were able to colonize concrete surfaces when supplied with moisture (95-100% relative humidity) and a nutrient source, and that fouling was affected by concrete water/cement ratio, surface roughness, and the presence of photocatalytically-activated cement added to inhibit microbial growth.